Composite

Part:BBa_K1921019

Designed by: Zhuozhi Chen   Group: iGEM16_TJUSLS_China   (2016-10-14)
Revision as of 07:20, 20 October 2016 by Zhizhi (Talk | contribs)

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LPP-OMPA+linker+PETase

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage

This part is a fusion protein of the Lpp-OmpA and PETase. PETase is a new protein enzyme found in bacteria which can decompose PET. Through this method, we can anchor PETase on the out membrane of Escherichia coli, then we can use this typical Escherichia coli to decompose PET. This is a way called whole cell catalysis. Using this method, we don’t need to purify the protein. In addition, prokaryote surface display system method is mature enough. Lpp-OmpA is frequently used to develop whole-cell biocatalysts and it consists of the first nine N-terminal amino acids of major E. coli lipoprotein (Lpp) bined with a transmembrane domain (46-159 aa) from outer membrane protein A (OmpA). We got its sequence from the genome of Escherichia coli str. K-12 substr.

Biology

PETase was found from a kind of microorganism(Ideonella sakaiensis 201-F6) living on PET as the main carbon source. It can degrade macromolecular polymers into monomers. PETase is the only enzyme found in bacteria which can degrade PET.
Surface expression of recombinant proteins was first described more than 30 years ago.The Lpp-OmpA consists of the first nine N-terminal amino acids of major E. coli lipoprotein (Lpp) bined with a transmembrane domain (46-159 aa) from outer membrane protein A (OmpA). We got its sequence from the genome of Escherichia coli str. K-12 substr.

Reference

[1] Ali Karami, Ali Mohamad Latifi*, and Samaneh Khodi,etal. 2014. Comparison of the Organophosphorus Hydrolase Surface Display Using InaVN and Lpp-OmpA Systems in Escherichia coli. J. Microbiol. Biotechnol. (2014),24(3), 379–385
[2] Joseph A. Francisco*, etal. 1992. Transport and anchoring of β-lactamase to the external surface of Escherichia coli.

Pre-expression

Tjuresults41.jpg
Figure 1.This is the pre-expression using E.coli BL21 at 37 ℃.

Tjuresults43.jpg
Figure 2. This is the pre-expression using E.coli BL21 at 16 ℃.

Tjuresults42.jpg
Figure 3. This is the pre-expression using E.coli BL21 at 25 ℃.

Surface display HPLC results

ProofTJU8.jpg
Figure 4. Relative enzyme activity of engineering bacteria E.coli(BL21)/pET22b(+)LAP when induced at 16℃.

[edit]
Categories
//awards/part_collection/2016
Parameters
None